The invention relates to a new transcriptional coactivator of steroid/nuclear receptors.
Steroids, thyroid hormones, vitamin D3, and retinoids are lipid-soluble small molecules which play a role in the control of cell differentiation, embryonic development, and homeostasis, as well as adult physiology. These molecules exert the majority of their effects on cells by interacting with specific receptors which, when bound by a specific ligand, affect transcription by interacting directly with chromatin. In addition, in their unliganded state, these receptors inhibit transcription of certain genes. The diverse biological effects of these molecules suggest that hormone actions are mediated by complex signaling. The hormone receptors comprise a large superfamily which displays substantial specificity in regulating gene expression (Beato et al. 1995. Cell 83: 851–857; Evans. 1988. Science 240: 889–895). These receptors share a common domain structure, including a N-terminal DNA-binding domain (DBD or C domain), which binds to specific DNA sequences, and a C-terminal ligand-binding domain (LBD or E domain), which binds to the cognate hormone. Retinoic acid receptors (RARs), thyroid hormone receptors (TRs), vitamin D3 receptor (VDR), peroxisomal proliferator activated receptors (PPARs), and several other orphan receptors form heterodimeric complexes with retinoid-X receptors (RXRs) (Yu et al., Cell 67: 1251–66, 1991; Kliewer et al., Nature 355:446–9, 1992; Willy et al., Genes & Development 2:1033–45, 1995). Such receptor heterodimers can bind to a broad range of response elements composed of two related half sites and activate target gene expression (Yu et al., Cell 67:1251–66, 1991; Kliewer et al., Nature 355:446–9, 1992; Umesono et al., Cell 65:1255–1266, 1991; Forman et al., Cell 81:541–550, 1996 Heyman et al., Cell 68:397–406, 1992).
Transcriptional activation by steroid/nuclear receptors is thought to involve at least two separate processes: derepression and activation (Mangelsdorf and Evans, Cell 83:841–850, 1995; Wong and Wolffe, Genes Dev.9:2696–711, 1995). Repression is effected in part by association of unliganded receptors with the nuclear receptor corepressors SMRT and N-CoR (Horlein et al., Nature 377:397–404, 1995; Chen and Evans, Nature 377:454–7, 1995). Ligand-binding triggers dissociation of these corepressors and recruitment of coactivators. Putative steroid/nuclear receptor coactivators have been identified, including: RIP-140 and RIP-160 (Cavailles et al., EMBO J. 14:3741–3751; Cavailles et al., Proc. Natl. Acad. Sci. USA 91:10009–13, 1994), ERAP-140 and ERAP-160 (Halachmi et al., Science 264:1455–8, 1994), TIF1 (Le Douarin et al., EMBO J. 14:2020–2033, 1995), steroid receptor coactivator-1 (SRC-1) (Kamei et al., Cell 85:403–14, 1996; Onate et al., Science 270:1354–1357, 1995), TRIP1/SUG1 (Lee et al., Nature 374:91–4, 1995), ARA70 (Yeh and Chang, Proc. Natl. Acad. Sci. USA 93:5517–21, 1996), transcriptional intermediate factor-2 (TIF2) (Voegel et al., EMBO J. 15:3667–3675, 1996), and CBP/p300 (Kamei et al., Cell 85:403–14, 1996; Chakravarti et al., Nature 383:99–103, 1996; Smith et al., Proc. Natl. Acad. Sci. USA 93:8884–8, 1996). Two of these potential coactivators, SRC-1 and TIF2, are related proteins and enhance transcriptional activation by several hormone receptors (Onate et al., Science 270:1354–1357, 1995; Voegel et al., EMBO J. 15:3667–3675, 1996; Smith et al., Proc. Natl. Acad. Sci. USA 93:8884–8, 1996; McInerney et al., Proc. Natl. Acad. Sci. USA 93:10069–73).
Hormone binding is thought to induce a conformational change in the receptor and, in turn, activate the C-terminal ligand-dependent activation function (AF-2) of the receptor (Mangeledorf et al., Cell 83:835–839, 1995). At the extreme C-terminus of the AF-2 domain, there are about 20 amino acids that form an amphipathic helix (Bourguet et al., Nature 375:377–82, 1995). This helix is referred to as the AF-2 activation domain (AF2-AD) (Renaud et al., Nature 378:681–9, 1995), TC, or T4 domain (Baniahmad et al., Mol. Cell. Bio. 15:76–86, 1995; Hollenberg and Evans, Cell 55:899–906, 1988). Deletion and several point mutations in this domain abolish the AF-2 function completely (Damm et al., Proc. Natl. Acad. Sci. USA 90:2989–2993, 1993; Schulman et al., Mol. Cell. Biol. 16:3807–13, 1995; Barettino et al., EMBO J. 13:3039–3049, 1994; Durand et al., EWBO J. 13:5370–5382,1994). The AF-2 domain can act alone as an activation domain (AD) when fused to a heterologous DNA binding domain (DBD) (Barettino et al., EMBO J. 13:3039–3049, 1994; Schulman et al., Proc. Natl. Acad. Sci. USA 92:8288–92, 1995). Comparison of the recent ligand binding domain (LBD) crystal structures of unliganded retinoid-X receptor α (RXRα; Bourguet et al., Nature 375:377–82, 1995) with liganded retinoic acid receptor γ (RARγ; Renaud et al., Nature 378:681–9, 1995) and liganded thyroid hormone receptor α (TRa; Wagner et al., Nature 378:690–697, 1995) reveals a striking difference in the relative position of the AF2-AD. It is proposed that, upon hormone binding, the AF2-AD rotates 180 degrees and forms part of the hormone binding surface, covering the ligand-binding cavity. The hydrophobic residues of the helix face the cavity, contacting the hydrophobic ligand, while the charged residues extend into the solvent, possibly mediating protein—protein interactions with coactivators (Bourguet et al., Nature 375:377–82, 1995; Renaud et al., Nature 378:681–9, 1995; Wagner et al., Nature 378:690–697, 1995). The AF2-AD domain has also been shown to be required for derepression by inducing dissociation of the corepressors (Chen and Evans, Nature 377:454–7, 1995; Baniahmad et al., Mol. Cell. Bio. 15:76–86, 1995). The discovery of novel molecules which are involved in hormone binding, transcriptional repression will allow for the modulation of responses to steroid hormones and, ultimately, will facilitate the modulation of cell differentiation, embryonic development, and homeostasis, as well as adult physiology.